2013 OAKRIDGE CONFERENCE, BALTIMORE, MD

Linan Song, Dandan Shan, Mingwei Zhao, Brian A. Pink, Kaitlin A. Minnehan, Lyndsey York, Melissa Gardel, Sean Sullivan, & David C. Duffy

Quanterix Corporation, 113 Hartwell Avenue, Lexington, MA 02421, USA

We describe a method capable of direct detection of genomic DNA and RNA extracted from bacteria or viruses at subfemtomolar concentrations with no necessary target amplification using polymerases. This approach includes first generating short nucleic acid fragments from large genomic DNA/RNA molecules either via random sonication or digestion using specific restriction enzymes. For DNA molecules, heat-denaturation is used to generate single-stranded fragments for capture. The method then involves the formation of hybridized complexes through capturing the fragmented target DNA/RNA on magnetic beads via sequence-specific capture probes, and hybridizing to multiple biotinylated detection probes, followed by labeling the resulting hybridized DNA/DNA or DNA/RNA complexes with streptavidin-conjugated beta-galactosidase. Signals from single labeled nucleic acid molecules on beads are generated by converting the substrate of beta-galactosidase, resorufin-β-D-galactopyranoside, into a fluorescent product in single molecule arrays (Simoa).  The resulting signal is reported as average number of enzymes per bead (AEB) that is correlated to the concentration of nucleic acid in a sample. We have demonstrated the detection of genomic DNA purified from S. aureus with an average limit of detection (LOD) of 0.07 fM, equivalent to 2,100 DNA molecules per 50 µL sample; and genomic RNA purified from Sendai virus with an average LOD of 0.09fM, equivalent to 2,700 RNA molecules per 50 µL sample. We also applied this Simoa technology to clinical and environmental samples for bacteria detection. An average sensitivity of 2,200 bacteria per 50 µL sample (0.074fM) and 1,300 bacteria per 50 µL sample (0.042fM) were obtained for S. aureus spiked into human whole blood and river water, respectively. The capability of detecting single enzyme molecules using Simoa enables the direct detection of target DNA and RNA without requiring target amplification, and provides a highly sensitive alternative approach to polymerase chain reactions (PCR) that is much less susceptible to carryover.